PFKM inhibits doxorubicin-induced cardiotoxicity by enhancing oxidative phosphorylation and glycolysis

Heart failure (HF) is a global pandemic which affects about 26 million people. PFKM (Phosphofructokinase, Muscle), catalyzing the phosphorylation of fructose-6-phosphate, plays a very important role in cardiovascular diseases. However, the effect of PFKM in glycolysis and HF remains to be elucidated. H9c2 rat cardiomyocyte cells were treated with doxorubicin (DOX) to establish injury models, and the cell viability, apoptosis and glycolysis were measured. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting were used for gene expression. DOX treatment significantly inhibited PFKM expression in H9c2 cells. Overexpression of PFKM inhibited DOX-induced cell apoptosis and DOX-decreased glycolysis and oxidative phosphorylation (OXPHOS), while silencing PFKM promoted cell apoptosis and inhibited glycolysis and OXPHOS in H9c2 cells. Moreover, PFKM regulated DOX-mediated cell viability and apoptosis through glycolysis pathway. Mechanism study showed that histone deacetylase 1 (HDAC1) inhibited H3K27ac-induced transcription of PFKM in DOX-treated cells and regulated glycolysis. PFKM could inhibit DOX-induced cardiotoxicity by enhancing OXPHOS and glycolysis, which might benefit us in developing novel therapeutics for prevention or treatment of HF.

PFKM downregulation promoted cell apoptosis and inhibited OXPHOS and glycolysis in H9c2 cells. Next, PFKM was silenced to further study its role. Silencing PFKM significantly inhibited cell viability ( Fig. 3A), promoted apoptosis (Fig. 3B,C), and inhibited PFKM, Bcl-2, but increased Bax (Fig. 3D,E). Silencing PFKM also suppressed OCR and ECAR (Fig. 3F,G), and decreased levels of ATP (Fig. 3H) and lactate (Fig. 3I). The results demonstrate that PFKM downregulation promoted cell apoptosis and inhibited OXPHOS and glycolysis in H9c2 cells. www.nature.com/scientificreports/ PFKM regulated DOX-mediated cell viability and apoptosis via glycolysis. To find out how PFKM regulates cell viability, glycolysis inhibitor, 2-DG, was introduced. Results showed that inhibition of glycolysis not only significantly promoted DOX-suppressed cell viability, but also abolished PFKM-overexpression-increased cell viability (Fig. 4A). TUNEL staining results showed that inhibition of glycolysis not only significantly increased DOX-increased cell apoptosis, but also abolished PFKM-overexpression-decreased cell apoptosis (Fig. 4B,C). The findings suggest that PFKM regulates DOX-mediated growth and apoptosis via the glycolysis pathway.

HDAC1 inhibited H3K27ac-induced transcription of PFKM in DOX-induced H9c2 cells. To fig-
ure out the mechanism by which PFKM is regulated, levels of H3K27ac and HDAC1 in DOX-treated H9c2 cells were measured. Results showed that DOX suppressed H3K27ac in a time-dependent manner, but increased the expression of HDAC1 (Fig. 5A,B). ChIP assay revealed that DOX treatment significantly suppressed the interaction between H3K27ac and the PFKM promoter (Fig. 5C). HDAC inhibitor, mocetinostat (MGCD), significantly promoted the interaction between H3K27ac and the PFKM promoter (Fig. 5D) and increased PKFM

Discussion
We revealed that DOX treatment significantly inhibited PFKM expression in H9c2 cells. Overexpressing of PFKM inhibited DOX-induced cell apoptosis and DOX-decreased glycolysis, while silencing PFKM promoted cell apoptosis and inhibited OXPHOS and glycolysis in H9c2 cells. Moreover, PFKM regulated DOX-mediated cell growth and apoptosis via glycolysis pathway. Data also supports that the expression of PFKM was suppressed by HDAC1 through regulating H3K27 acetylation. For the first time, we show that HDAC1-mediated PFKM down-regulation promoted cell apoptosis and inhibited OXPHOS in H9c2 cells through regulating glycolysis (Fig. 6F), which may provide novel directions for new drug development. Glycolysis regulated energy metabolism 22 . Glucose is converted into pyruvate, NADH, and ATP by glycolysis 23 . Glycolysis involves in many biological and pathological processes. For example, glycolysis promotes tumor growth 24 . Preclinical studies demonstrate that some small molecules such as 3-bromopyruvate suppresses cancer via targeting glycolysis 25 . Another study showed that Smad4 depletion in podocytes protects mice from glomerulosclerosis 26 . Inhibition of aerobic glycolysis causes depression of cardiac excitability and can lead to Ca 2+ alternant in cardiac tissue 27 . Increased glycolysis is the earliest energy metabolic change during heart failure with preserved ejection fraction 28 . Other studies have demonstrated that glycolysis affects sarcoplasmic reticulum (SR) function and SR Ca 2+ release not only through generation of ATP but also through direct interactions of www.nature.com/scientificreports/ glycolytic intermediates and products with the Ca 2+ release channel itself 27 . Moreover, hyperglycemia enhances ipilimumab-induced cardiotoxicity through mechanisms mediated by MyD88 and NLRP3 signaling 7 , suggesting that targeting the MyD88/NLRP3 signaling may inhibit ipilimumab-induced cardiotoxicity in patients with cardiovascular diseases. In this study, we demonstrated that suppressing glycolysis remarkably ameliorated the effect of PFKM on DOX-mediated cell growth and apoptosis. These results reveal a very important role of glycolysis in regulating DOX-mediated cell growth and apoptosis and improve our knowledge of the role of glucose homeostasis in the reduction of doxorubicin cardiotoxicity. PFK catalyzes the rate-limiting phosphorylation of fructose-6-phosphate and sustains a high rate of glycolysis 8 . It has 3 isoforms: platelet (PFKP), muscle (PFKM), and liver (PFKL) 29 . PFKM gene has 24 exons 30 . PFK deficiency belongs to glycogen storage disease characterized by weakness with spasms and cramping on exercise 30 . Ristow et al. have reported that deficiency of PFKM results in insulin resistance, contributing to diabetes 31 . Studies also indicate that PFKM plays a very important role in cardiovascular diseases. For instance, Garcia et al. indicated that PFK deficiency causes a cardiac and hematological disorder 12 . Two-month-old PFKM knockout mice developed cardiac hypertrophy and evident cardiomegaly with age 12 . Preclinical studies correlate high levels of IL-1β to a greater risk of cardiovascular diseases; the underlying mechanism of cardiotoxicity involves the dysfunction of mitochondrial metabolism 32 . Therefore, pharmacological inhibition of IL-1β could be a promising approach for the treatment of cardiovascular diseases. In this study, PFKM downregulation also inhibited OXPHOS in H9c2 cells. However, the role of IL-1β in PFKM-induced glucose and mitochondrial metabolism in DOX-treated H9c2 cells need further investigation. Our findings indicate a very important role of PFKM in regulating the proliferation of cardiomyoblasts and cardiotoxicity and improve our knowledge of FPKM in the pathogenesis of HF.
Protein acetylation is the process that the acetyl group is transferred to a polypeptide chain 33 . Acetylation alters protein function 33 . Protein acetylation plays a very important role in diverse physiological processes 34,35 . H3K27ac involves in the higher activation of transcription 36 . It is elevated in mammary cancer and administration of H3K27ac inhibitor repressed tumor formation 37 . Felice et al. demonstrated that hypoacetylation of H3K27 involves in intestinal inflammation 38 . H3K27ac also involves in cardiovascular diseases. For example, a study indicated that H3K27ac acetylation status regulates phenotypic response in HF 39 . Papait et al. reported that H3K27ac was decreased in mice with transverse aortic constriction 40 . Our findings suggested that HDAC1 significantly decreased the level of H3K27ac to suppress the transcription of PFKM and regulate OXPHOS and glycolysis. These findings indicated the significance of HDAC1/H3K27ac/PFKM axis in cardiotoxicity and HF, which may benefit the study of HF and cardiovascular diseases. Keep in mind that only in vitro cell experiments were used in this study. Future studies with animals or even clinical samples will definitely supply more meaningful data. Nevertheless, our study revealed new roles of PFKM and glycolysis in HF.  TdT-mediated dUTP nick-end labeling (TUNEL) staining. TUNEL staining was used for apoptosis 41 .

Extracellular flux (XF) analysis.
Twenty-four hours after treatment, glycolysis and mitochondrial respiration levels were monitored by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using a Seahorse XF24 Extracellular Flux Analyzer 42 . Briely, cells digested to a density of 1 × 10 4 / well, were seeded in XF-24 culture plates (Agilent Technologies, Santa Clara, CA, USA, 100777-004), and were then placed in an incubator of 37 °C and 5% CO 2 for 24 h. Around 1 h before detection, cells were shifted into an incubator without CO 2 , and culture medium was replaced by XF Base Medium (Agilent Technologies, Santa Clara, CA, USA, 103335-100). Subsequently, 1 μM oligomycin (ATP synthase inhibitor) was added into "A" well of Seahorse gauging plate, 1.5 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; uncoupler) was supplemented into "B" well and then mixture of antimycin A (complex III inhibitor; 0.5 μM) & rotenone (complex I inhibitor; 0.5 μM) was instilled into "C" well using Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA, 103015-100). Using a Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA), cellular OCR was monitored. In addition, the cells were treated sequentially with 1 μM of glucose, 1 μM of oligomycin, and 0.5 μM of 2-DG (the glycolytic inhibitor) at time points for measurement of ECAR.

Measurement of lactate and ATP.
The cells were seeded in 96-well plates at 3.5 × 10 3 cells per well. After overnight incubation at 37 ℃, 5% CO 2 , the complete medium was changed to fresh DMEM (50 μl/well). After 24 h, the supernatant of cells was collected by centrifugation. Then, according to the manufacturer's instructions, the lactate release was determined using Lactic Acid assay kit (Nanjing Jiancheng Bioengineering Institute, China). ATP content was measured with the ATP assay kit (Nanjing Jiancheng Bioengineering Institute, China), as per the manufacturer's protocol. In brief, cells were seeded in the 6-well plate for 12-24 h. Then cells were harvested by using 200-300 μl lysis buffer and vortexed for 1 min. The supernatant was mixed with detection solution and then analysis for ATP concentration was normalized to the corresponding total protein amounts from each sample. Immunoblotting. Cell lysates were extracted using radioimmunoprecipitation assay buffer (JRDUN Biotechnology, Co., Ltd., Shanghai, China). Total protein concentration in each sample was measured using a Lowry protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equivalent quantities (25 μg) of protein were separated by 10 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Sigma-Aldrich), followed by blocking in fat-free milk overnight at 4˚C. The membranes were incubated with primary antibodies, including anti-PFKM antibody (ab154804, dilution 1:1000, Abcam), anti-Bcl-2 antibody (ab182858, dilution 1:2000, Abcam), anti-Bax antibody (ab32503, dilution 1:10,000, Abcam), anti-H3K27ac antibody (ab177178, dilution 1:10,000, Abcam), anti-HDAC1 antibody (10,197- Chromatin immunoprecipitation (ChIP). ChIP analysis was performed as previously described 43 .

Reverse transcription-polymerase chain reaction (RT-PCR). Total
Briefly, cells with 2 μM DOX treatment were cross-linked in 1% formaldehyde, and the DNA was sonicated into a size range of 200-1000 base pairs using a Bioruptor Sonicator (Diagenode) for five cycles of 3 son/3 s off.

Data availability
We confirm that all data generated or analyzed during this study are available from the corresponding author on reasonable request.